The Effect Of ClC-3 On Angiotensin ? Induced Oxidative Stress In Endothelial Cells And The Underlying Mechanisms

The integrity of the structure and functions of endothelium is critical for cardiovascular system. Under normal condition, endothelial cells play an important role in regulating the vessel functions by modulating the reactivity of the vessels to mechanical stretch and transmitter through releasing relaxing and contracting factors. Accumulating evidence indicated that the impaired endothelium- dependent relaxation due to the decrease of NO level or bioavailability is the early marker of cardiovascular disorders, like hypertension, atherosclerosis and hyperlipidemia. Increased reactive oxidative stress(ROS) production is one of the primary causes leading to endothelium dysfunction.Angiotensin II(Ang II) is the major bioactive peptide in the renin–angiotensin system(RAS), plays a major role in the regulation of vascular function and structure. Ang II regulates vascular cell production of ROS through NADPH oxidase(NOX), including Nox1, Nox2, Nox4, and Nox5. Activation of these NOX leads to ROS generation, which in turn influences many downstream signaling targets of Ang II, including Rho A/Rho kinases, transcription factors and tyrosine kinases. Activation of these redox-sensitive pathways regulates vascular cell growth, inflammation and senescence.ClC-3, a member of the Cl C voltage-gated chloride(Cl-) channel family, is ubiquitously expressed in almost all mammalian cells. ClC-3 could function as volume-regulated Cl- channels or Cl-/H+ antiporter, play a role in regulation of physiology and pathology activities including intracellular PH, cell volume, proliferation, differentiation, migration, apoptosis. It was reported that superoxide induced by Ang II flux across the cell membrane was mediated by ClC-3 channel in pulmonary micro-vascular endothelial cells. The trans-membrane flux of extracellular O2- can be significantly inhibited by ClC-3 si RNA or the Cl- channel blocker 4,4-diisothiocyanostilbene-2,2-disulfonic acid(DIDS). ClC-3 absence inhibited the NADPH oxidase-derived ROS production induced by tumor necrosis factor-?(TNF-?) and interleukin-1?(IL-1?) in vascular smooth muscle cells. In addition, compare with wild type mice, the activity of NADPH oxidase was declined in neutrophile granulocyte of ClC-3 knock out mice, accompanied with the function of trans-membrane transfer, migration and phagocytose. In our previous study, we found that the activity of ClC-3 volume-regulated Cl- channels is increased in the process of hypertension and ClC-3 deficiency inhibited Ang II-induced EPC apoptosis via suppressing ROS generation derived from NADPH oxidase. Based on these data, ClC-3 plays a role in the regulation of ROS production in several cell types. However, the mechanism of ClC-3 regulating NADPH oxidase/ROS generation is still unclear.Considering the essential role of ROS production in the development of cardiovascular diseases,the aim of our present study is to investigate the effects and mechanism of ClC-3 on NADPH oxidase and ROS generation in endothelial cells.PART I: The effect of ClC-3 on Angiotensin? induced oxidative stress in endothelial cellsBy using adenovirus transfection, immunofluorescence, SA-beta-galactosidase staining, we investigated the effects of ClC-3 on Ang II-induced oxidative stress in endothelial cells or aortas of Ang II-induced hypertensive mice. Results were as follows:1. Ang II increases superoxide anion production in HUVEC. Knockdown of ClC-3 does not affect superoxide anion production in the basal level, but attenuates superoxide anion production increase after Ang II stimulation, overexpression of ClC-3further enhances superoxide anion production in basal level and after Ang II stimulation.(n=6, **P<0.01 vs. CON, ##P<0.01 vs. Ang II+CON)2. Silience of ClC-3 does not affect NOX activity in the basal level, but attenuates NOX activity increase after Ang II stimulation, whereas overexpression of ClC-3 enhances significantly NADPH oxidase activity in basal level and after Ang II stimulation.(n=4,*P <0.05 vs CON, #P<0.05 vs. Ang II)3. Furthermore, we establish an animal model of hypertension by treating the ClC-3 knockout and wild type mouse with the subcutaneous implantable pump that infused with Ang II, then test ROS production of aortas. Ang II significantly inceases ROS production in aortas of WT mice. ROS production is significantly greater in aortas from in WT- Ang II than those in KO-Ang II and there is no statistic difference between KO-Con and KO-Ang II.(n=3, **P<0.01 vs. WT-Con, ##P<0.01 vs. WT-Ang II)4. For further study effect of ClC-3 on Ang ?induced oxidative stress in endothelial cells, we use ?-Galactosidase Staining Kit to test Ang ?induced HUVEC senescence. Ang ?increases significantly ?-Galactosidase activity which indicates cell senescence, knockdown of ClC-3 does not affect ?-Galactosidase activity in the basal level, but inhibits ?-Galactosidase activity increase after Ang II stimulation, whereas overexpression of ClC-3 increases significantly ?-Galactosidase oxidase activity in basal level and after Ang II stimulation. Representative photomicrographs show senescence associated ?-galactosidase(SA-?-Gal)positive cells with different treatments.Summary:1. ClC-3 increases O2- production in endothelial cells, ClC-3 increases ROS production in endothelial cells mainly through enhancing NADPH oxidase activity.2. ClC-3 increases vascular ROS generation. Downregulation of ClC-3 decrease ROS production during hypertension.3. ClC-3 potentiated Ang II-induced aging, Downregulation of ClC-3 inhibits cell senescence.PART II: The mechanism of ClC-3 on NADPH oxidase in endothelial cellsBy using adenovirus transfection, co-immunoprecipitation, western blot, we investigated the mechanism of ClC-3 on Ang II-induced NOX activity increase in endothelial cells. Results were as follows:1. In basal level, knockdown of ClC-3 has no effect on the expression of NOX2 and p22 phox, whereas the expression of NOX2 and p22 phox are increased by overexpression ClC-3. Ang II increased the expression of NOX2 and p22 phox, which can be attenuated by knockdown ClC-3, and further enhanced by overexpression ClC-3(n=5, *P<0.05 vs. CON, #P<0.05 vs. Ang II). Knockdown or overexpression of ClC-3 did not alter the expression of p47 phox, p67 phox and Rac1.(n=3)2. ClC-3 interacts with p22 phox, p47 phox, p67 phox and Rac1 respectively. No interaction between ClC-3 and NOX2 is observed.3. Ang II significantly increases p22phox/p47 phox and p67phox/p47 phox interaction in HUVEC. ClC-3 potentiates p22 pohx interaction with p47 phox. Representative immunoblot of p47 phox in p22 phox immunoprecipitates in cell lysates. ClC-3 potentiates p47 pohx interaction with p67 phox. Representative immunoblot of p47 phox in p67 phox immunoprecipitates in cell lysates.(n=4, *P <0.05 vs. CON, #P<0.05 vs. Ang II)4. Ang II has no effect on the total expression of p47 phox and p67 phox in HUVEC(figure2-1), but significantly increases the expression of p47 phox and p67 phox on cell membrane. As a result, Ang II increases translocation of p47 phox and p67 phox from cytoplasm to cell membrane in HUVEC. Knockdown of ClC-3 does not affect expression of p47 phox and p67 phox on cell membrane in the basal level, but attenuates p47 phox and p67 phox increase after Ang II stimulation, overexpression of ClC-3further enhances p47 phox and p67 phox expression in basal level and after Ang II stimulation.(n=5,*P<0.05 vs. CON, #P<0.05 vs. Ang II)5. Ang II significantly increases p47 phox phosphorylation. Knockdown of ClC-3 decreases Ang II-induced p47 phox phosphorylation and do not affect p47 phox phosphorylation in basal level. Overexpression of ClC-3 enhances Ang II-induced p47 phox phosphorylation.(n=4,*P<0.05 vs. CON, #P<0.05 vs. Ang II)6. Ang II significantly increases p38-MAPK phosphorylation. Knockdown of ClC-3 decreases Ang II-induced p38-MAPK phosphorylation and do not affect p38-MAPK phosphorylation in basal level. Overexpression of ClC-3 enhances Ang II-induced p38-MAPK phosphorylation.(n=3,*P<0.05 vs. CON, #P<0.05 vs. Ang II)7. The increased p47 phox phosphorylation in ClC-3 overexpressed cells was also abolished by blockage of p38 signaling pathway.(n=6,**P<0.01 vs. Ang II, ##P<0.01 vs. Ang II+Ad-Cl C 3)Summary:1. Overexpression of ClC-3 increases the expression of NOX2 and p22 phox, enhances p47 phox interacting with p22 phox p67phox, and elevates the translocation of p47 phox and p67 phox, whereas knockdown of ClC-3 abolishes these effects.2. Knockdown of ClC-3 decreases Ang II-induced p47 phox and p38-MAPK phosphorylation. Overexpression of ClC-3 increases Ang II-induced p47 phox and p38-MAPK phosphorylation.3. ClC-3 increases p47 phox phosphorylation through p38-MAPK pathway.Conclusion1. ClC-3 increases NADPH oxidase activity and oxidase stress in endothelial cells.2. ClC-3 enhances the assembly of NADPH oxidase through increasing the expression of NOX2 and p22 phox, enhancing p47 phox and p67phox translocation to cell membrane in HUVEC, then increases NADPH oxidase activity.3. ClC-3 increases p47 phox phosphorylation through p38-MAPK pathway, then enhances the interactions of p22 phox with p47 phox, and p67 phox with p47 phox, increases the translocation of p47 phox and p67 phox to cell membrane, thus promotes the formation of NADPH oxidase complex.